The high affinity receptor for IgE ("E-receptor") on mast cells plays a central role in immediate hypersensitivity. Reaction of receptor-bound IgE with polyvalent antigen clusters the receptors and this stimulates a variety of cellular responses characteristic of allergic and inflammatory events. We study the molecular mechanisms by which E-receptors generate such responses. During the past year, we have principally explored an apparent exception to the "kinetic proofreading" mechanism which we had previously shown to exist in the mast cell line we use to study the E-receptor. When receptor-initiated signaling is subject to kinetic proofreading, then the degree to which the system proceeds through sequential steps of a signaling cascade is governed by the lifetime of the initial ligand: receptor interaction. Earlier studies revealed evidence for this mechanism. However,we also observed that even ligands that interact relatively weakly can stimulate certain late events; in particular, the synthesis of mRNAs coding for various mediators. Much of our effort during the past year has been to explore the basis for this apparent paradox. We have postulated (and explored by mathematical modeling) the possibility that certain late signals are responding to intracellular messengers that are not associated with the receptor:ligand complex thereby forming a branch that is no longer constrained by kinetic proofreading. In particular we have monitored intracellular Ca2+ as a candidate for such a messenger. We observed that its increase is effectively stimulated by the low affinity ligand and that this is sufficient to promote the synthesis of the mRNA we are monitoring. We have further documented that the rise in Ca2+ is inddeed the critical event leading to the transcription of the gene we monitored. Using an alternative assay we were also able to monitor the translation of the mRNA and the secretion of protein product, the chemokine MCP-1. A novel fining was that whereas aggregation of the receptor and the rise in Ca2+ were required for both the transcription and secretion they were not required for translation of the mRNA. Finally, we have completed the studies in which we are trying to understand the basis of the large effects of cytoskeletal perturbing agents on the initial events stimulated by the E-receptor. The similarity of E-receptors to other receptors of the immune system (e.g. the clonotypic receptors on T and B lymphocytes), make it likely that the significance of our studies extends beyond the IgE/mast cell system.